AU707061B2 - Production of gamma linolenic acid by a delta6-desaturase - Google Patents

Production of gamma linolenic acid by a delta6-desaturase Download PDF

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AU707061B2
AU707061B2 AU46735/96A AU4673596A AU707061B2 AU 707061 B2 AU707061 B2 AU 707061B2 AU 46735/96 A AU46735/96 A AU 46735/96A AU 4673596 A AU4673596 A AU 4673596A AU 707061 B2 AU707061 B2 AU 707061B2
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plant
gla
desaturase
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Georges L Freyssinet
Michael Nuccio
Andrew N Nunberg
Avutu S Reddy
Terry L Thomas
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Bayer SAS
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Rhone Poulenc Agrochimie SA
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Description

WO 96/21022 PCT/IB95/01167 -1- 1 PRODUCTION OF GAMMA LINOLENIC ACID BY A A6-DESATURASE Linoleic acid (18:2) (LA) is transformed into gamma linolenic acid (18:3) (GLA) by the enzyme A6-desaturase. When this enzyme, or the nucleic acid encoding it, is transferred into LA-producing cells, GLA is produced. The present invention provides nucleic acids comprising the A6-desaturase gene. More specifically, the nucleic acids comprise the promoters, coding regions and termination regions of the A6-desaturase genes. The present invention is further directed to recombinant constructions comprising a A6-desaturase coding region in functional combination with heterologous regulatory sequences.
The nucleic acids and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms.
Unsaturated fatty acids such as linoleic
(C
18
A
9 12 and a-linolenic (C 8
A
912 1 5 s) acids are essential dietary constituents that cannot be synthesized by vertebrates since vertebrate cells can introduce double bonds at the A 3 position of fatty acids but cannot introduce additional double bonds between the A' double bond and the methyl-terminus of the fatty acid chain. Because they are precursors of other products, linoleic and a-linolenic acids are essential fatty acids, and are usually obtained from plant sources. Linoleic acid can be converted by mammals into y-linolenic acid (GLA, CA 6 9 12 which can in turn be converted to arachidonic acid a critically SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -2- 1 important fatty acid since it is an essential precursor of most prostaglandins.
The dietary provision of linoleic acid, by virtue of its resulting conversion to GLA and arachidonic acid, satisfies the dietary need for GLA and arachidonic acid. However, a relationship has been demonstrated between consumption of saturated fats and health risks such as hypercholesterolemia, atherosclerosis and other clinical disorders which correlate with susceptibility to coronary disease, while the consumption of unsaturated fats has been associated with decreased blood cholesterol concentration and reduced risk of atherosclerosis.
The therapeutic benefits of dietary GLA may result from GLA being a precursor to arachidonic acid and thus subsequently contributing to prostaglandin synthesis. Accordingly, consumption of the more unsaturated GLA, rather than linoleic acid, has potential health benefits. However, GLA is not present in virtually any commercially grown crop plant.
Linoleic acid is converted into GLA by the enzyme A6-desaturase. A6-desaturase, an enzyme of more than 350 amino acids, has a membrane-bound domain and an active site for desaturation of fatty acids.
When this enzyme is transferred into cells which endogenously produce linoleic acid but not GLA, GLA is produced. The present invention, by providing the gene encoding A6-desaturase, allows the production of transgenic organisms which contain functional A6desaturase and which produce GLA. In addition to SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -3- 1 allowing production of large amounts of GLA, the present invention provides new dietary sources of GLA.
The present invention is directed to isolated A6-desaturase genes. Specifically, the isolated genes comprises the A6-desaturase promoters, coding regions, and termination regions.
The present invention is further directed to expression vectors comprising the A6-desaturase promoter, coding region and termination region.
Yet another aspect of this invention is directed to expression vectors comprising a A6desaturase coding region in functional combination with heterologous regulatory regions, i.e. elements not derived from the a6-desaturase gene.
Cells and organisms comprising the vectors of the present invention, and progeny of such organisms, are also provided by the present invention.
A further aspect of the present invention provides isolated bacterial a6-desaturase. An isolated plant A6-desaturase is also provided.
Yet another aspect of this invention provides a method for producing plants with increased gamma linolenic acid content.
A method for producing chilling tolerant plants is also provided by the present invention.
Fig. 1 depicts the hydropathy profiles of the deduced amino acid sequences of Synechocystis A6desaturase (Panel A) and al2-desaturase (Panel B).
Putative membrane spanning regions are indicated by solid bars. Hydrophobic index was calculated for a SUBSTITUTE SHEET (RULE 26)
I
WO 96/21022 PCT/I95/01167 -4- 1 window size of 19 amino acid residues [Kyte, et al.
(1982) J. Molec. Biol. 157].
Fig. 2 provides gas liquid chromatography profiles of wild type (Panel A) and transgenic (Panel B) Anabaena.
Fig. 3 is a diagram of maps of cosmid cSyl3 and Csy7 with overlapping regions and subclones.
The origins of subclones of Csy75, Csy75-3.5 and Csy7 are indicated by the dashed diagonal lines.
Restriction sites that have been inactivated are in parentheses.
Fig. 4 provides gas liquid chromatography profiles of wild type (Panel A) and transgenic (Panel B) tobacco.
Fig. 5A depicts the DNA sequence of a A-6 desaturase cDNA isolated from borage.
Fig. 5B depicts the protein sequence of the open reading frame in the isolated borage a-6 desaturase cDNA. Three amino acid motifs characteristic of desaturases are indicated and are, in order, lipid box, metal box 1, and metal box 2.
Fig. 6 is a dendrogram showing similarity of the borage A6-desaturase to other membrane-bound desaturases. The amino acid sequence of the borage A6-desaturase was compared to other known desaturases using Gene Works (IntelliGenetics). Numerical values correlate to relative phylogenetic distances between subgroups compared.
Fig. 7 is a restriction map of 221.A6.NOS and 121.A6.NOS. In 221.A6.NOS, the remaining portion SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 1 of the plasmid is pBI221 and in 121.A6.NOS, the remaining portion of the plasmid is pBI121.
Fig. 8 provides gas liquid chromatography profiles of mock transfected (Panel A) and 221.A6.NOS transfected (Panel B) carrot cells. The positions of 18:2, 18:3 a, and 18:3 y(GLA) are indicated.
Fig. 9 provides gas liquid chromatography profiles of an untransformed tobacco leaf (Panel A) and a tobacco leaf transformed with 121.A6.NOS. The positions of 18:2, 18:3 a, 18:3y(GLA), and 18:4 are indicated.
Fig. 10 provides gas liquid chromotography profiles for untransformed tobacco seeds (Panel A) and seeds of tobacco transformed with 121.A6.NOS. The positions of 18:2, 18:3a and 18:3(GLA) are indicated.
The present invention provides isolated nucleic acids encoding A6-desaturase. To identify a nucleic acid encoding A6-desaturase, DNA is isolated from an organism which produces GLA. Said organism can be, for example, an animal cell, certain fungi Mortierella), certain bacteria (e.g.
Synechocystis) or certain plants (borage, Oenothera, currants). The isolation of genomic DNA can be accomplished by a variety of methods well-known to one of ordinary skill in the art, as exemplified by Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY. The isolated DNA is fragmented by physical methods or enzymatic digestion and cloned into an appropriate vector, e.g. a bacteriophage or cosmid vector, by any of a variety of well-known methods which can be found SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/I95/01167 -6- 1 in references such as Sambrook et al. (1989).
Expression vectors containing the DNA of the present invention are specifically contemplated herein. DNA encoding A6-desaturase can be identified by gain of function analysis. The vector containing fragmented DNA is transferred, for example by infection, transconjugation, transfection, into a host organism that produces linoleic acid but not GLA. As used herein, "transformation" refers generally to the incorporation of foreign DNA into a host cell.
Methods for introducing recombinant DNA into a host organism are known to one of ordinary skill in the art and can be found, for example, in Sambrook et al.
(1989). Production of GLA by these organisms gain of function) is assayed, for example by gas chromatography or other methods known to the ordinarily skilled artisan. Organisms which are induced to produce GLA, i.e. have gained function by the introduction of the vector, are identified as expressing DNA encoding A6-desaturase, and said DNA is recovered from the organisms. The recovered DNA can again be fragmented, cloned with expression vectors, and functionally assessed by the above procedures to define with more particularity the DNA encoding a6desaturase.
As an example of the present invention, random DNA is isolated from the cyanobacteria Synechocystis Pasteur Culture Collection (PCC) 6803, American Type Culture Collection (ATCC) 27184, cloned into a cosmid vector, and introduced by transconjugation into the GLA-deficient cyanobacterium SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/fB95/01167 -7- 1 Anabaena strain PCC 7120, ATCC 27893. Production of GLA from Anabaena linoleic acid is monitored by gas chromatography and the corresponding DNA fragment is isolated.
The isolated DNA is sequenced by methods well-known to one of ordinary skill in the art as found, for example, in Sambrook et al. (1989).
In accordance with the present invention, DNA molecules comprising A6-desaturase genes have been isolated. More particularly, a 3.588 kilobase (kb) DNA comprising a A6-desaturase gene has been isolated from the cyanobacteria Synechocystis. The nucleotide sequence of the 3.588 kb DNA was determined and is shown in SEQ ID NO:1. Open reading frames defining potential coding regions are present from nucleotide 317 to 1507 and from nucleotide 2002 to 3081. To define the nucleotides responsible for encoding A6desaturase, the 3.588 kb fragment that confers a6desaturase activity is cleaved into two subfragments, each of which contains only one open reading frame.
Fragment ORF1 contains nucleotides 1 through 1704, while fragment ORF2 contains nucleotides 1705 through 3588. Each fragment is subcloned in both forward and reverse orientations into a conjugal expression vector (AM542, Wolk et al. [1984] Proc. Natl. Acad. Sci. USA 81, 1561) that contains a cyanobacterial carboxylase promoter. The resulting constructs ORF1(F), ORF1(R), ORF2(F) and ORF2(R)] are conjugated to wildtype Anabaena PCC 7120 by standard methods (see, for example, Wolk et al. (1984) Proc. Natl. Acad. Sci. USA 81, 1561). Conjugated cells of Anabaena are SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -8- 1 identified as Neo" green colonies on a brown background of dying non-conjugated cells after two weeks of growth on selective media (standard mineral media BG11N containing 30yg/ml of neomycin according to Rippka et al., (1979) J. Gen Microbiol. 111, 1).
The green colonies are selected and grown in selective liquid media (BG11N with 15pg/ml neomycin). Lipids are extracted by standard methods Dahmer et al., (1989) Journal of American Oil Chemical Society 66, 543) from the resulting transconjugants containing the forward and reverse oriented ORF1 and ORF2 constructs.
For comparison, lipids are also extracted from wildtype cultures of Anabaena and Synechocystis. The fatty acid methyl esters are analyzed by gas liquid chromatography (GLC), for example with a Tracor-560 gas liquid chromatograph equipped with a hydrogen flame ionization detector and a capillary column. The results of GLC analysis are shown in Table 1.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 1 Table 1: Occurrence and of C18 fatty acids in wild-type transgenic cyanobacteria I I _9
SOURCE
18:0 18:1 18:2 "lR: Snl Anabaena (wild type) Anabaena ORF1(F) Anabaena ORF1(R) 8:3 18:4 *1 Anabaena ORF2(F) 77 Anabaena ORF2(R) I I I i I Synechocystis (wild type)
I
As assessed by GLC analysis, GLA deficient Anabaena gain the function of GLA production when the construct containing ORF2 in forward orientation is introduced by transconjugation. Transconjugants containing constructs with ORF2 in reverse orientation to the carboxylase promoter, or ORF1 in either orientation, show no GLA production. This analysis demonstrates that the single open reading frame (ORF2) within the 1884 bp fragment encodes a6-desaturase.
The 1884 bp fragment is shown as SEQ ID NO:3. This is substantiated by the overall similarity of the hydropathy profiles between a6-desaturase and A12desaturase [Wada et al. (1990) Nature 347] as shown in Fig. 1 as and respectively.
Also in accordance with the present invention, a cDNA comprising a A6-desaturase gene from 3 borage (Boraqo officinalis) has been isolated. The nucleotide sequence of the 1.685 kilobase (kb) cDNA SUBSTITUTE SHEET (RULE 26)
I
WO 96/21022 PCT/IB95/01167 1 was determined and is shown in Fig. 5A (SEQ ID NO: 4).
The ATG start codon and stop codon are underlined.
The amino acid sequence corresponding to the open reading frame in the borage delta 6-desaturase is shown in Fig. 5B (SEQ ID NO: Isolated nucleic acids encoding a6desaturase can be identified from other GLA-producing organisms by the gain of function analysis described above, or by nucleic acid hybridization techniques using the isolated nucleic acid which encodes Synechocystis or borage a6-desaturase as a hybridization probe. Both genomic and cDNA cloning methods are known to the skilled artisan and are contemplated by the present invention. The hybridization probe can comprise the entire DNA sequence disclosed as SEQ. ID NO:1 or SEQ. ID NO:4, or a restriction fragment or other DNA fragment thereof, including an oligonucleotide probe. Methods for cloning homologous genes by cross-hybridization are known to the ordinarily skilled artisan and can be found, for example, in Sambrook (1989) and Beltz et al. (1983) Methods in Enzymoloqy 100, 266.
In another method of identifying a delta 6desaturase gene from an organism producing GLA, a cDNA library is made from poly-A' RNA isolated from polysomal RNA. In order to eliminate hyper-abundant expressed genes from the cDNA population, cDNAs or fragments thereof corresponding to hyper-abundant cDNAs genes are used as hybridization probes to the cDNA library. Non hybridizing plaques are excised and the resulting bacterial colonies are used to inoculate SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -11- 1 liquid cultures and sequenced. For example, as a means of eliminating other seed storage protein cDNAs from a cDNA library made from borage polysomal RNA, cDNAs corresponding to abundantly expressed seed storage proteins are first hybridized to the cDNA library. The "subtracted" DNA library is then used to generate expressed sequence tags (ETSs) and such tags are used to scan a data base such as GenBank to identify potential desaturates.
Transgenic organisms which gain the function of GLA production by introduction of DNA encoding adesaturase also gain the function of octadecatetraeonic acid (18:4A 9 15 s) production.
Octadecatetraeonic acid is present normally in fish oils and in some plant species of the Boraginaceae family (Craig et al. [1964] J. Amer. Oil Chem. Soc.
41, 209-211; Gross et al. [1976] Can.. J. Plant Sci.
56, 659-664). In the transgenic organisms of the present invention, octadecatetraenoic acid results from further desaturation of a-linolenic acid by &6desaturase or desaturation of GLA by The 359 amino acids encoded by ORF2, i.e.
the open reading frame encoding Synechocystis a6desaturase, are shown as SEQ. ID NO:2. The open reading frame encoding the borage A6-desaturase is shown in SEQ ID NO: 5. The present invention further contemplates other nucleotide sequences which encode the amino acids of SEQ ID NO:2 and SEQ ID NO: 5. It is within the ken of the ordinarily skilled artisan to identify such sequences which result, for example, from the degeneracy of the genetic code. Furthermore, SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -12- 1 one of ordinary skill in the art can determine, by the gain of function analysis described hereinabove, smaller subfragments of the fragments containing the open reading frames which encode a6-desaturases.
The present invention contemplates any such polypeptide fragment of a6-desaturase and the nucleic acids therefor which retain activity for converting LA to GLA.
In another aspect of the present invention, a vector containing a nucleic acid of the present invention or a smaller fragment containing the promoter, coding sequence and termination region of a A6-desaturase gene is transferred into an organism, for example, cyanobacteria, in which the A6-desaturase promoter and termination regions are functional.
Accordingly, organisms producing recombinant A6desaturase are provided by this invention. Yet another aspect of this invention provides isolated A6desaturase, which can be purified from the recombinant organisms by standard methods of protein purification.
(For example, see Ausubel et al. [1987] Current Protocols in Molecular Biology, Green Publishing Associates, New York).
Vectors containing DNA encoding A6desaturase are also provided by the present invention.
It will be apparent to one of ordinary skill in the art that appropriate vectors can be constructed to direct the expression of the A6-desaturase coding sequence in a variety of organisms. Replicable expression vectors are particularly preferred.
Replicable expression vectors as described herein are SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -13- 1 DNA or RNA molecules engineered for controlled expression of a desired gene, i.e. the a 6 -desaturase gene. Preferably the vectors are plasmids, bacteriophages, cosmids or viruses. Shuttle vectors, e.g. as described by Wolk et al. (1984) Proc. Natl.
Acad. Sci. USA, 1561-1565 and Bustos et al. (1991) J.
Bacteriol. 174, 7525-7533, are also contemplated in accordance with the present invention. Sambrook et al. (1989), Goeddel, ed. (1990) Methods in Enzvmology 185 Academic Press, and Perbal (1988) A Practical Guide to Molecular Cloning, John Wiley and Sons, Inc., provide detailed reviews of vectors into which a nucleic acid encoding the present &6-desaturase can be inserted and expressed. Such vectors also contain nucleic acid sequences which can effect expression of nucleic acids encoding a6-desaturase. Sequence elements capable of effecting expression of a gene product include promoters, enhancer elements, upstream activating sequences, transcription termination signals and polyadenylation sites. Both constitutive and tissue specific promoters are contemplated. For transformation of plant cells, the cauliflower mosaic virus (CaMV) 35S promoter and promoters which are regulated during plant seed maturation are of particular interest. All such promoter and transcriptional regulatory elements, singly or in combination, are contemplated for use in the present replicable expression vectors and are known to one of ordinary skill in the art. The CaMV 355 promoter is described, for example, by Restrepo et al. (1990) SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -14- 1 Plant Cell 2, 987. Genetically engineered and mutated regulatory sequences are also contemplated.
The ordinarily skilled artisan can determine vectors and regulatory elements suitable for expression in a particular host cell. For example, a vector comprising the promoter from the gene encoding the carboxylase of Anabaena operably linked to the coding region of A6-desaturase and further operably linked to a termination signal from Synechocystis is appropriate for expression of A6-desaturase in cyanobacteria. "Operably linked" in this context means that the promoter and terminator sequences effectively function to regulate transcription. As a further example, a vector appropriate for expression of A6-desaturase in transgenic plants can comprise a seed-specific promoter sequence derived from helianthinin, napin, or glycinin operably linked to the A6-desaturase coding region and further operably linked to a seed termination signal or the nopaline synthase termination signal. As a still further example, a vector for use in expression of A 6desaturase in plants can comprise a constitutive promoter or a tissue specific promoter operably linked to the A 6-desaturase coding region and further operably linked to a constitutive or tissue specific terminator or the nopaline synthase termination signal.
In particular, the helianthinin regulatory elements disclosed in applicant's copending
U.S.
Application Serial No. 682,354, filed April 8, 1991 and incorporated herein by reference, are contemplated SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 1 as promoter elements to direct the expression of the A6-desaturase of the present invention.
Modifications of the nucleotide sequences or regulatory elements disclosed herein which maintain the functions contemplated herein are within the scope of this invention. Such modifications include insertions, substitutions and deletions, and specifically substitutions which reflect the degeneracy of the genetic code.
Standard techniques for the construction of such hybrid vectors are well-known to those of ordinary skill in the art and can be found in references such as Sambrook et al. (1989), or any of the myriad of laboratory manuals on recombinant DNA technology that are widely available. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments. It is further contemplated in accordance with the present invention to include in the hybrid vectors other nucleotide sequence elements which facilitate cloning, expression or processing, for example sequences encoding signal peptides, a sequence encoding KDEL, which is required for retention of proteins in the endoplasmic reticulum or sequences encoding transit peptides which direct A6-desaturase to the chloroplast. Such sequences are known to one of ordinary skill in the art. An optimized transit peptide is described, for example, by Van den Broeck et al. (1985) Nature 313, 358.
Prokaryotic and eukaryotic signal sequences are SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -16- 1 disclosed, for example, by Michaelis et al. (1982) Ann. Rev. Microbiol. 36, 425.
A further aspect of the instant invention provides organisms other than cyanobacteria or plants which contain the DNA encoding the A6-desaturase of the present invention. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989).
A variety of plant transformation methods are known. The a6-desaturase gene can be introduced into plants by a leaf disk transformation-regeneration procedure as described by Horsch et al. (1985) Science 227, 1229. Other methods of transformation, such as protoplast culture (Horsch et al. (1984) Science 223, 496; DeBlock et al. (1984) EMBO J. 2, 2143; Barton et al. (1983) Cell 32, 1033) can also be used and are within the scope of this invention. In a preferred embodiment plants are transformed with Agrobacteriumderived vectors. However, other methods are available to insert the a6-desaturase genes of the present invention into plant cells. Such alternative methods include biolistic approaches (Klein et al. (1987) Nature 327, 70), electroporation, chemically-induced DNA uptake, and use of viruses or pollen as vectors.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -17- 1 When necessary for the transformation method, the a6-desaturase genes of the present invention can be inserted into a plant transformation vector, e.g. the binary vector described by Bevan (1984) Nucleic Acids Res. 12, 8111. Plant transformation vectors can be derived by modifying the natural gene transfer system of Acrobacterium tumefaciens. The natural system comprises large Ti (tumor-inducing)-plasmids containing a large segment, known as T-DNA, which is transferred to transformed plants. Another segment of the Ti plasmid, the vir region, is responsible for T-DNA transfer. The T-DNA region is bordered by terminal repeats. In the modified binary vectors the tumor-inducing genes have been deleted and the functions of the vir region are utilized to transfer foreign DNA bordered by the T-DNA border sequences. The T-region also contains a selectable marker for antibiotic resistance, and a multiple cloning site for inserting sequences for transfer. Such engineered strains are known as "disarmed" A. tumefaciens strains, and allow the efficient transformation of sequences bordered by the T-region into the nuclear genomes of plants.
Surface-sterilized leaf disks are inoculated with the "disarmed" foreign DNA-containing A.
tumefaciens, cultured for two days, and then transferred to antibiotic-containing medium.
Transformed shoots are selected after rooting in medium containing the appropriate antibiotic, transferred to soil and regenerated.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -18- 1 Another aspect of the present invention provides transgenic plants or progeny of these plants containing the isolated DNA of the invention. Both monocotyledenous and dicotyledenous plants are contemplated. Plant cells are transformed with the isolated DNA encoding a6-desaturase by any of the plant transformation methods described above. The transformed plant cell, usually in a callus culture or leaf disk, is regenerated into a complete transgenic plant by methods well-known to one of ordinary skill in the art Horsch et al. (1985) Science 227, 1129). In a preferred embodiment, the transgenic plant is sunflower, oil seed rape, maize, tobacco, peanut or soybean. Since progeny of transformed plants inherit the DNA encoding A6-desaturase, seeds or cuttings from transformed plants are used to maintain the transgenic plant line.
The present invention further provides a method for providing transgenic plants with an increased content of GLA. This method includes introducing DNA encoding A6-desaturase into plant cells which lack or have low levels of GLA but contain LA, and regenerating plants with increased GLA content from the transgenic cells. In particular, commercially grown crop plants are contemplated as the transgenic organism, including, but not limited to, sunflower, soybean, oil seed rape, maize, peanut and tobacco.
The present invention further provides a method for providing transgenic organisms which contain GLA. This method comprises introducing DNA SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -19- 1 encoding a6-desaturase into an organism which lacks or has low levels of GLA, but contains LA. In another embodiment, the method comprises introducing one or more expression vectors which comprise DNA encoding al2-desaturase and A6-desaturase into organisms which are deficient in both GLA and LA. Accordingly, organisms deficient in both LA and GLA are induced to produce LA by the expression of A12-desaturase, and GLA is then generated due to the expression of a6desaturase. Expression vectors comprising DNA encoding al2-desaturase, or A12-desaturase and a6desaturase, can be constructed by methods of recombinant technology known to one of ordinary skill in the art (Sambrook et al., 1989) and the published sequence of al2-desaturase (Wada et al [1990] Nature (London) 347, 200-203. In addition, it has been discovered in accordance with the present invention that nucleotides 2002-3081 of SEQ. ID NO:1 encode cyanobacterial a12-desaturase. Accordingly, this sequence can be used to construct the subject expression vectors. In particular, commercially grown crop plants are contemplated as the transgenic organism, including, but not limited to, sunflower, soybean, oil seed rape, maize, peanut and tobacco.
The present invention is further directed to a method of inducing chilling tolerance in plants.
Chilling sensitivity may be due to phase transition of lipids in cell membranes. Phase transition temperature depends upon the degree of unsaturation of fatty acids in membrane lipids, and thus increasing the degree of unsaturation, for example by introducing SUBSTITUTE SHEET (RULE 26) Q:\OPER\JMS\46735-96.127 7/5/99 A6-desaturase to convert LA to GLA, can induce or improve chilling resistance.
Accordingly, the present method comprises introducing DNA encoding A6-desaturase into a plant cell, and regenerating a plant with improved chilling resistance from said transformed plant cell. In a preferred embodiment, the plant is a sunflower, soybean, oil seed rape, maize, peanut or tobacco plant.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
The following examples further illustrate the present invention.
S S S S o o* WO 96/21022 PCT/IB95/01167 -21- EXAMPLE 1 Strains and Culture Conditions Synechocystis (PCC 6803, ATCC 27184), Anabaena (PCC 7120, ATCC 27893) and Synechococcus (PCC 7942, ATCC 33912) were grown photoautotrophically at 0 C in BG11N+ medium (Rippka et al. [1979] J. Gen.
Microbiol. 111, 1-61) under illumination of incandescent lamps (60pE.m 2 Cosmids and plasmids were selected and propagated in Escherichia coli strain DH5a on LB medium supplemented with antibiotics at standard concentrations as described by Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, New York.
SUBS(UE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -22- 1 EXAMPLE 2 Construction of Synechocystis Cosmid Genomic Library Total genomic DNA from Synechocystis (PCC 6803) was partially digested with Sau3A and fractionated on a sucrose gradient (Ausubel et al.
[1987] Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York). Fractions containing 30 to 40 kb DNA fragments were selected and ligated into the dephosphorylated BamHI site of the cosmid vector, pDUCA7 (Buikema et al. [1991] J. Bacteriol. 173, 1879-1885). The ligated DNA was packaged in vitro as described by Ausubel et al. (1987), and packaged phage were propagated in E.
coli DH5a containing the Aval and Eco4711 methylase helper plasmid, pRL528 as described by Buikema et al.
(1991). A total of 1152 colonies were isolated randomly and maintained individually in twelve 96-well microtiter plates.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -23- 1 EXAMPLE 3 Gain-of-Function Expression of GLA in Anabaena Anabaena (PCC 7120), a filamentous cyanobacterium, is deficient in GLA but contains significant amounts of linoleic acid, the precursor for GLA (Figure 2; Table The Synechocystis cosmid library described in Example 2 was conjugated into Anabaena (PCC 7120) to identify transconjugants that produce GLA. Anabaena cells were grown to mid-log phase in BG11N+ liquid medium and resuspended in the same medium to a final concentration of approximately 2x10' cells per ml. A mid-log phase culture of E.
coli RP4 (Burkardt et al. [1979] J. Gen. Microbiol.
114, 341-348) grown in LB containing ampicillin was washed and resuspended in fresh LB medium. Anabaena and RP4 were then mixed and spread evenly on BG11N+ plates containing 5% LB. The cosmid genomic library was replica plated onto LB plates containing 50 yg/ml kanamycin and 17.5 ig/ml chloramphenicol and was subsequently patched onto BG11N+ plates containing Anabaena and RP4. After 24 hours of incubation at 0 C, 30 yg/ml of neomycin was underlaid; and incubation at 300C was continued until transconjugants appeared.
Individual transconjugants were isolated after conjugation and grown in 2 ml BG11N+ liquid medium with 15 Ag/ml neomycin. Fatty acid methyl esters were prepared from wild type cultures and cultures containing pools of ten transconjugants as follows. Wild type and transgenic cyanobacterial SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB901167 -24- 1 cultures were harvested by centrifugation and washed twice with distilled water. Fatty acid methyl esters were extracted from these cultures as described by Dahmer et al. (1989) J. Amer. Oil. Chem. Soc. 66, 543- 548 and were analyzed by Gas Liquid Chromatography (GLC) using a Tracor-560 equipped with a hydrogen flame ionization detector and capillary column (30 m x 0.25 mm bonded FSOT Superox II, Alltech Associates Inc., IL). Retention times and co-chromatography of standards (obtained from Sigma Chemical Co.) were used for identification of fatty acids. The average fatty acid composition was determined as the ratio of peak area of each C18 fatty acid normalized to an internal standard.
Representative GLC profiles are shown in Fig. 2. C18 fatty acid methyl esters are shown.
Peaks were identified by comparing the elution times with known standards of fatty acid methyl esters and were confirmed by gas chromatography-mass spectrometry. Panel A depicts GLC analysis of fatty acids of wild type Anabaena. The arrow indicates the migration time of GLA. Panel B is a GLC profile of fatty acids of transconjugants of Anabaena with pAM542+1.8F. Two GLA producing pools (of 25 pools representing 250 transconjugants) were identified that produced GLA. Individual transconjugants of each GLA positive pool were analyzed for GLA production; two independent transconjugants, AS13 and AS75, one from each pool, were identified which expressed significant levels of GLA and which contained cosmids, cSyl3 and respectively (Figure The cosmids overlap SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 1 in a region approximately 7.5 kb in length. A 3.5 kb NheI fragment of cSy75 was recloned in the vector pDUCA7 and transferred to Anabaena resulting in gainof-function expression of GLA (Table 2).
Two NheI/Hind III subfragments (1.8 and 1.7 kb) of the 3.5 kb Nhe I fragment of cSy75-3.5 were subcloned into "pBLUESCRIPT" (Stratagene) (Figure 3) for sequencing. Standard molecular biology techniques were performed as described by Maniatis et al. (1982) and Ausubel et al. (1987). Dideoxy sequencing (Sanger et al. [1977] Proc. Natl. Acad. Sci. USA 74, 5463- 5467) of pBS1.8 was performed with "SEQUENASE" (United States Biochemical) on both strands by using specific oligonucleotide primers synthesized by the Advanced DNA Technologies Laboratory (Biology Department, Texas A M University). DNA sequence analysis was done with the GCG (Madison, WI) software as described by Devereux et al. (1984) Nucleic Acids Res. 12, 387-395.
Both NheI/HindIII subfragments were transferred into a conjugal expression vector, AM542, in both forward and reverse orientations with respect to a cyanobacterial carboxylase promoter and were introduced into Anabaena by conjugation.
Transconjugants containing the 1.8 kb fragment in the forward orientation (AM542-1.8F) produced significant quantities of GLA and octadecatetraenoic acid (Figure 2; Table Transconjugants containing other constructs, either reverse oriented 1.8 kb fragment or forward and reverse oriented 1.7 kb fragment, did not produce detectable levels of GLA (Table 2).
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -26- 1 Figure 2 compares the C18 fatty acid profile of an extract from wild type Anabaena (Figure 2A) with that of transgenic Anabaena containing the 1.8 kb fragment of cSy75-3.5 in the forward orientation (Figure 2B). GLC analysis of fatty acid methyl esters from AM542-1.8F revealed a peak with a retention time identical to that of authentic GLA standard. Analysis of this peak by gas chromatography-mass spectrometry (GC-MS) confirmed that it had the same mass fragmentation pattern as a GLA reference sample.
Transgenic Anabaena with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCTlIB95/01167 -27- STable 2 Composition of C18 Fatty Acids in Wild Type and Transgenic Cyanobacteria Fatty Acid (t) Strain 18:0 18: 1 18:2 18.3 1 8 3 18.4 Wild Type Synechocystis 13.6 4.5 54.5 27.3 (sp.PCC6803) Anabaena 2.9 24.8 37.1 35.2 (sp .PCC7l2O) Synechococcus 20.6 79.4 (sp.PCC7942) Anabaena Transconj ugantB 3.8 24.4 22.3 9.1 27.9 12.5 cSy75-3.5 4.3 27.6 18.1 3.2 40.4 6.4 pM4 .F4.2 13.9 12.1 19.1 25.4 25.4 pAM542 1.8R 7.7 23.1 38.4 30.8 pAM542 1-7F 2.8 27.8 36.1 33.3 pAM542 1.7R 2.8 25.4 42.3 29.6 Synechococcus Trans formant s pAM854 27.8 72.2 pAM854 _-M 2 4.0 43.2 46.0 pAM8S4 18.2 81.8 pAM854 2 42.7 25.3 19.5 -16.5 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3(a), linolenic acid; 18 3 y-linolenic acid; 18:4, octadecatetraenoic acid SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -28- 1 EXAMPLE 4 Transformation of Synechococcus with a6 and 412 Desaturase Genes A third cosmid, cSy7, which contains a a12desaturase gene, was isolated by screening the Synechocystis genomic library with a oligonucleotide synthesized from the published Synechocystis A12desaturase gene sequence (Wada et al. [1990] Nature (London) 347, 200-203). A 1.7 kb Aval fragment from this cosmid containing the Al2-desaturase gene was identified and used as a probe to demonstrate that cSyl3 not only contains a A6-desaturase gene but also a al2-desaturase gene (Figure Genomic Southern blot analysis further showed that both the a6-and A12desaturase genes are unique in the Synechocvstis genome so that both functional genes involved in C18 fatty acid desaturation are linked closely in the Synechocystis genome.
The unicellular cyanobacterium Synechococcus (PCC 7942) is deficient in both linoleic acid and GLA(3). The A12 and a6-desaturase genes were cloned individually and together into pAM854 (Bustos et al.
[1991] J. Bacteriol. 174, 7525-7533), a shuttle vector that contains sequences necessary for the integration of foreign DNA into the genome of Svnechococcus (Golden et al. [1987] Methods in Enzvmol. 153, 215- 231). Synechococcus was transformed with these gene constructs and colonies were selected. Fatty acid methyl esters were extracted from transgenic Synechococcus and analyzed by GLC.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -29- 1 Table 2 shows that the principal fatty acids of wild type Synechococcus are stearic acid (18:0) and oleic acid Synechococcus transformed with pAM854-A12 expressed linoleic acid (18:2) in addition to the principal fatty acids. Transformants with pAM854-A6 and A12 produced both linoleate and GLA (Table These results indicated that Synechococcus containing both al2- and A6-desaturase genes has gained the capability of introducing a second double bond at the a12 position and a third double bond at the A6 position of C18 fatty acids. However, no changes in fatty acid composition was observed in the transformant containing pAM854-a6, indicating that in the absence of substrate synthesized by the a12 desaturase, the A6-desaturase is inactive. This experiment further confirms that the 1.8 kb NheI/HindIII fragment (Figure 3) contains both coding and promoter regions of the Svnechocystis A6desaturase gene. Transgenic Synechococcus with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB9/01167 1 EXAMPLE Nucleotide Sequence of A6-Desaturase The nucleotide sequence of the 1.8 kb fragment of cSy75-3.5 including the functional A6desaturase gene was determined. An open reading frame encoding a polypeptide of 359 amino acids was identified (Figure A Kyte-Doolittle hydropathy analysis (Kyte et al. [1982] J. Mol. Biol. 157, 105- 132) identified two regions of hydrophobic amino acids that could represent transmembrane domains (Figure 1A); furthermore, the hydropathic profile of the A6desaturase is similar to that of the Al2-desaturase gene (Figure 1B; Wada et al.) and A9-desaturases (Thiede et al. [1986] J. Biol. Chem. 261, 13230- 13235). However, the sequence similarity between the Synechocystis A6- and al2-desaturases is less than at the nucleotide level and approximately 18% at the amino acid level.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -31- 1 EXAMPLE 6 Transfer of Cyanobacterial A'-Desaturase into Tobacco The cyanobacterial a-desaturase gene was mobilized into a plant expression vector and transferred to tobacco using Agrobacterium mediated gene transfer techniques. To ensure that the transferred desaturase is appropriately expressed in leaves and developing seeds and that the desaturase gene product is targeted to the endoplasmic reticulum or the chloroplast, various expression cassettes with Synechocystis a-desaturase open reading frame (ORF) were constructed. Components of these cassettes include: a 35S promoter or seed specific promoter derived from the sunflower helianthinin gene to drive a 6 -desaturase gene expression in all plant tissues or only in developing seeds respectively, (ii) a putative signal peptide either from carrot extensin gene or sunflower helianthinin gene to target newly synthesized a 6 -desaturase into the ER, (iii) an ER lumen retention signal sequence (KDEL) at the COOHterminal of the A'-desaturase ORF, and (iv) an optimized transit peptide to target A6 desaturase into the chloroplast. The 35S promoter is a derivative of pRTL2 described by Restrepo et al. (1990). The optimized transit peptide sequence is described by Van de Broeck et al. (1985). The carrot extensin signal peptide is described by Chen et al (1985) EMBO J. 9, 2145.
Transgenic tobacco plants were produced containing a chimeric cyanobacterial desaturase gene, SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -32- 1 comprised of the Synechocvstis a 6 desaturase gene fused to an endoplasmic reticulum retention sequence (KDEL) and extensin signal peptide driven by the CaMV promoter. PCR amplifications of transgenic tobacco genomic DNA indicate that the A6 desaturase gene was incorporated into the tobacco genome. Fatty acid methyl esters of leaves of these transgenic tobacco plants were extracted and analyzed by Gas Liquid Chromatography (GLC). These transgenic tobacco accumulated significant amounts of GLA (Figure 4).
Figure 4 shows fatty acid methyl esters as determined by GLC. Peaks were identified by comparing the elution times with known standards of fatty acid methyl ester. Accordingly, cyanobacterial genes involved in fatty acid metabolism can be used to generate transgenic plants with altered fatty acid compositions.
suBsllUlx StEET (RULE 26) WO 96/21022 PCT/IB95/01167 -33- 1 EXAMPLE 7 Construction of Borage cDNA library Membrane bound polysomes were isolated from borage seeds 12 days post pollination (12 DPP) using the protocol established for peas by Larkins and Davies (1975 Plant Phys. 55:749-756). RNA was extracted from the polysomes as described by Mechler (1987 Methods in Enzymology 152:241-248, Academic Press).
Poly-A+ RNA was isolated from the membrane bound polysomal RNA by use of Oligotex-dT beads (Qiagen). Corresponding cDNA was made using Stratagene's ZAP cDNA synthesis kit. The cDNA library was constructed in the lambda ZAP II vector (Stratagene) using the lambda ZAP II vector kit. The primary library was packaged in Gigapack II Gold packaging extract (Stratagene). The library was used to generate expressed sequence tags (ESTs), and sequences corresponding to the tags were used to scan the GenBank database.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -34- 1 EXAMPLE 8 Hybridization Protocol Hybridization probes for screening the borage cDNA library were generated by using random primed DNA synthesis as described by Ausubel et al (1994 Current Protocols in Molecular Biology, Wiley Interscience, and corresponded to previously identified abundantly expressed seed storage protein cDNAs. Unincorporated nucleotides were removed by use of a G-50 spin column (Boehringer Manheim). Probe was denatured for hybridization by boiling in a water bath for 5 minutes, then quickly cooled on ice. Filters for hybridization were prehybridized at 60 0 C for 2-4 hours in prehybridization solution (6XSSC [Maniatis et al 1984 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory], 1X Denharts Solution, 0.05% sodium pyrophosphate, 100 Ag/ml denatured salmon sperm DNA). Denatured probe was added to the hybridization solution (6X SSC, 1X Denharts solution, 0.05% sodium pyrophosphate, 100 ig/ml denatured salmon sperm DNA) and incubated at 60 0 C with agitation overnight.
Filters were washed in 4x, 2x, and Ix SET washes for minutes each at 60 0 C. A 20X SET stock solution is 3M NaCl, 0.4 M Tris base, 20 mM Na 2 EDTA-2H 2 0. The 4X SET wash was 4X SET, 12.5 mM PO4, pH 6.8 and 0.2% SDS.
The 2X SET wash was 2X SET, 12.5 mM PO,, pH 6.8 and 0.2% SDS. The IX SET wash was IX SET, 12.5 mM PO 4 pH 6.8 and 0.2% SDS. Filters were allowed to air dry and were then exposed to X-ray film for 24 hours with intensifying screens at -80 0
C.
SUBSTITUTE SHEET (RULE 26) 0 WO 96/21022 PCT/IB95/01167 1 EXAMPLE 9 Random sequencing of cDNAs from a borage seed (12 DPP) membrane-bound polysomal library The borage cDNA library was plated at low density (500 pfu on 150 mm petri dishes). Highly prevalent seed storage protein cDNAs were "subtracted" by screening with the previously identified corresponding cDNAs. Non-hybridizing plaques were excised using Stratagene's excision protocol and reagents. Resulting bacterial colonies were used to inoculate liquid cultures and were either sequenced manually or by an ABI automated sequencer. Each cDNA was sequenced once and a sequence tag generated from 200-300 base pairs. All sequencing was performed by cycle sequencing (Epicentre). Over 300 ESTs were generated. Each sequence tag was compared to GenBank database by BLASTX computer program and a number of lipid metabolism genes, including the A6-desaturase were identified.
Database searches with a cDNA clone designated mbp-65 using BLASTX with the GenBank database resulted in a significant match to the Svnechocystis A6-desaturase. It was determined 2 however, that this clone was not a full length cDNA.
A full length cDNA was isolated using mbp-65 to screen the borage membrane-bound polysomal library. The sequence of the isolated cDNA was determined (Fig. SEQ ID NO:4) and the protein sequence of the open reading frame (Fig. 5B, SEQ ID NO:5) was compared to other known desaturases using Geneworks SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -36- 1 (IntelligGenetics) protein alignment program (Fig. 2).
This alignment indicated that the cDNA was the borage A6-desaturase gene.
Although similar to other known plant desaturases, the borage delta 6-desaturase is distinct as indicated in the dendrogram shown in Fig. 6.
Furthermore, comparison of the amino acid sequences characteristic of desaturases, particularly those proposed to be involved in metal binding (metal box 1 and metal box illustrates the differences between the borage delta 6-desaturase and other plant desaturases (Table 3).
The borage delta 6-desaturase is distinguished from the cyanobacterial form not only in over all sequence (Fig. 6) but also in the lipid box, metal box 1 and metal box 2 amino acid motifs (Table As Table 3 indicates, all three motifs are novel in sequence. Only the borage delta 6-desaturase metal box 2 shown some relationship to the Synechocystis delta-6 desaturase metal box 2.
In addition, the borage delta 6-desaturase is also distinct from another borage desaturase gene, the delta-12 desaturase. P1-81 is a full length cDNA that was identified by EST analysis and shows high similarity to the Arabidopsis delta-12 desaturase (Fad A comparison of the lipid box, metal box 1 and metal box 2 amino acid motifs (Table 3) in borage delta 6 and delta-12 desaturases indicates that little homology exists in these regions. The placement of the two sequences in the dendrogram in Fig. 6 indicates how distantly related these two genes are.
SUBSTITUTE SHEET (RULE 26) Table 3. Comparison of common amino acid motifs in membrane-bound desaturases Amino Acid Motif Desaturaso Lipid Box Metal Box 1 Metal Box 2 Borage A' WIGHDAGH Synechocystis A6 NVGHDANH Arab. chioroplast VLGHDCGH Rice
VLGIIDCGH
Glycine chioroplast A5 VLGHDCGH Arab. fad3 (A" 5
VLGHDCGH
Brassica fad3 (A" 5
VLGHDCGH
Borage A" 2 (P1-81)* VIAHECGH Arab. fad2 (A1 2
VIAHECGH
Arab. chioroplast A12 VIGHDCAH Glycine plastid A1 2
VIGHDCAH
Spinach plastidial n-6 VIGHDCAM Synechocystis A1 2
VVGHDCGH
Anabaena A1 2
VLGHDCGH
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21) 22) 22) 22) 22) 22) 23) 23) 24) 24) 24) 24) (SEQ. ID. NO: 17) (SEQ. ID. NO: 18) (SEQ. ID. NO: 19) *Pl-81 is a full length cDNA which was identified Arbidopsis A12 desaturase (fad2) by EST analysis and shows high similarity to the WO 96/21022 PCT/IB95/01167 -38- 1 EXAMPLE Construction of 222.1A6NOS for transient and expression The vector pBI221 (Jefferson et al. 1987 EMBO J. 6:3901-3907) was prepared for ligation by digestion with BamHI and EcoICR I (Promega) which excises the GUS coding region leaving the 35S promoter and NOS terminator intact. The borage A 6-desaturase cDNA was excised from the Bluescript plasmid (Stratagene) by digestion with BamHI and XhoI. The XhoI end was made blunt by use of the Klenow fragment.
This fragment was then cloned into the BamHI/EcoICR I sites of pBI221, yielding 221.A 6 NOS (Fig. In 221.A 6 .NOS, the remaining portion (backbone) of the restriction map depicted in Fig. 7 is pBI221.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -39- 1 EXAMPLE 11 Construction of 121.A 6 .NOS for stable transformation The vector pBIl21 (Jefferson et al. 1987 EMBO J. 6:3901-3907) was prepared for ligation by digestion with BamHI and EcoICR I (Promega) which excises the GUS coding region leaving the 35S promoter and NOS terminator intact. The borage A 6-desaturase cDNA was excised from the Bluescript plasmid (Stratagene) by digestion with BamHI and XhoI. The XhoI end was made blunt by use of the Klenow fragment.
This fragment was then cloned into the BamHI/EcoICR
I
sites of pBIl21, yielding 121.I16NOS (Fig. In 121.A 6 .NOS, the remaining portion (backbone) of the restriction map depicted in Fig. 7 is pBIl21.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 1 EXAMPLE 12 Transient Expression All work involving protoplasts was performed in a sterile hood. One ml of packed carrot suspension cells were digested in 30 mls plasmolyzing solution g/1 KC1, 3.5 g/1 CaC12-H 2 0, 10mM MES, pH 5.6 and 0.2 M mannitol) with 1% cellulase, 0.1% pectolyase, and 0.1% dreisalase overnight, in the dark, at room temperature. Released protoplasts were filtered through a 150 pm mesh and pelleted by centrifugation (100x g, 5 min.) then washed twice in plasmolyzing solution. Protoplasts were counted using a double chambered hemocytometer. DNA was transfected into the protoplasts by PEG treatment as described by Nunberg and Thomas (1993 Methods in Plant Molecular Biology and Biotechnology, B.R. Glick and J.E. Thompson, eds.
pp. 241-248) using 106 protoplasts and 50-70 ug of plasmid DNA (221.A6.NOS). Protoplasts were cultured in 5 mls of MS media supplemented with 0.2M mannitol and 3 pm 2,4-D for 48 hours in the dark with shaking.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -41- 1 EXAMPLE 13 Stable transformation of tobacco 121.A 6 .NOS plasmid construction was used to transform tobacco (Nicotiana tabacum cv. xanthi) via Agrobacterium according to standard procedures (Horsh et al., 1985 Science 227: 1229-1231; Bogue et al., 1990 Mol. Gen. Genet. 221:49-57), except that initial transformants were selected on 100 ug/ml kanamycin.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -42- 1 EXAMPLE 14 Preparation and analysis of fatty acid methyl esters (FAMEs) Tissue from transfected protoplasts and transformed tobacco plants was frozen in liquid nitrogen and lyophilized overnight. FAMEs were prepared as described by Dahmer et al (1989 J. Amer.
Oil Chem. Soc. 66:543-548). In some cases, the solvent was evaporated again, and the FAMEs were resuspended in ethyl acetate and extracted once with deionized water to remove any water soluble contaminants. The FAMEs were analyzed by gas chromatography (GC) on a J&W Scientific DB-wax column m length, 0.25 mm ID, 0.25 um film).
An example of a transient assay is shown in Fig. 8 which represents three independent transfections pooled together. The addition of the borage A6-desaturase cDNA corresponds with the appearance of gamma linolenic acid (GLA) which is one of the possible products of A6-desaturase.
Figures 9 and 10 depict GC profiles of the FAMES derived from leaf and seed tissue, respectively, of control and transformed tobacco plants. Figure 9A provides the profile of leaf tissue of wild-type tobacco (xanthi); Figure 9B provides the profile of leaf tissue from a tobacco plant transformed with the borage A-6 desaturase under the transcriptional control of the 35S CaMV promoter (pBI 121AENOS) Peaks correspond to 18:2, 18:3y (GLA), 18:3a and 18:4 (octadecanonic acid). Figure 10A shows the GC profile of seeds of a wild-type tobacco; Figure 10B shows the SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -43- 1 profile of seed tissue of a tobacco plant transformed with pBI 121 6 ANOS. Peaks correspond to 18:2, 18:3y(GLA) and 18:3a.
The relative distribution of the Ci fatty acids in control and transgenic tobacco seeds is shown in Table 4.
TABLE 4 Fatty Acid Xanthi pBI121A 6N
OS
18:0 4.0% 18:1 13% 13% 18:2 82% 82% 18:3y(GLA) 2.7% 18:3c 0.82% 1.4% The foregoing results demonstrate that GLA is incorporated into the triacylglycerides of transgenic tobacco leaves and seeds containing the borage A6-desaturase.
SUBSTITUTE SHEET (RULE 26) WO 96/21022 PCT/IB95/01167 -44- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Rhone-Poulenc Agrochimie (ii) TITLE OF INVENTION: PRODUCTION OF GAMMA LINOLENIC ACID BY A DELTA 6-DESATURASE (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: Scully, Scott, Murphy Presser STREET: 400 Garden City Plaza CITY: Garden City STATE: New York COUNTRY: United States ZIP: 11530 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION
NUMBER:
FILING DATE: 30-DEC-1994
CLASSIFICATION:
(viii) ATTORNEY/AGENT
INFORMATION:
NAME: Presser, Leopold REGISTRATION NUMBER: 19,827 REFERENCE/DOCKET NUMBER: 8383ZYXW (ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: (516) 742-4343 TELEFAX: (516) 742-4366 TELEX: 230 901 SANS UR INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3588 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 96/21022 WO 96/ 1022PCTIB9S/01 167 (ix) FEATURE: NAME/KEY: CDS LOCATION: 2002. .3081 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCTAGCCACC
TCCCCGCATT
CACCTTGCCA
TGCGGCTTTG
TCAGGAAATT
GGATGATccG
AGGCGCAGTG
ACCCCAACCC
CCTGCGGGAG
GATGATTTTT
CGCGTTGTAT
AAAGTCCCCC
GATTGGTATT
TTTGGATGCG
GAGCATGGCC
GGATACAGAT
GGATGCCCGC
GGTGGCCACC
CCCTAc3CCTG
AGTATTTGAA
GGCGGCCCTG
CCTAGCCACC
CCAAAAGTCT
GGAATTATTG
TGCCCTAGAG
AGTGACGATG
CGCATTGTTA
GACCACGTTA
GGCAATCAGG
GTCATTCACC
AGCCGAATGT
GTGAATAATT
AAGACCAAAC
TATCAGCGGT
CTGGCCACCT
TTTTCCGTGG
GATATCATCA
TGTTATGCCC
GCCAAGTTAC
ATTATTGAAG
AATCGTTTCT
CTAGAAAGAA
AGCGACGACA
CCAGTGGTGT
TTTGAAACGG
GGGGGCAAAA
TTAATCACTC
GATTTCGTTC
GGTACCCATC
CAACTTTGGC
CCTTGAATTT
ATCGTTTGTT
GTTTGAGTGT
CGATCGGGCA
AAGACCATCC
TGATCTATTA
TAACGTTGCA
GGCGATCGCC
ATGTCCAACA
TCATCTACGT
GCATGATTAC
AAGTATTCAC
TACTGAATGA
CCGATCGCCA
AGTTAATTCA
TGCATACGGC
CGTTGGCCTG
CCGTTAACTT
TGCGTTGCCA
TGCTTTGTCC
TTTTGGGCAA
CTAACCATCC
CCCTCTATCT
rCGACTCTGG
GATCGCCCCG
GGCCATTCTG
CAACCATGCC
TTCCGCCCTG
ATTGCGTTTG
CTGGCTCAAT
CCTACCGGCC
ATCTGGGGAC
TTGGCGCAAA
GGTGATATGG
TTCCATTGAT
CGGGGCCGGT
AGTGGTGATG
TTTCATCCTT
TCACATCATC
CCAGGGCCAT
CCGCTCCCTG
CGCCAATATC
GGAAATTGGC
GGATGCCCAG
GGCGGAATTG
CGGCATGACC
CTTTGCCGAC
%.GAACGGGGT
%.GACGTGTTG
CGCCACTGCT
ACCCAGGCCC
CTGGGTAAAC
GCGGCCCCGA
TTTGACCAGA
TTACCCCTGG
CACAGTGAAA
CATTTAATAG
TTTTCCAAAC
GTGGTGTTGT
CAACATATTG
GGCAAGGAAG
ATGATCGCCG
GGCAGTCGCT
ATTTGTGGGC
GAA.ATTGTGG
GGGGTGCCCG
AACCGAGCCG
CTAACTGCCA
TTTAGCCTGT
GCCACCTATT
GATGATTTGC
CAATTGGTTA
GGCAAAACCA
rATTTAACCA GATCCTCTGG I~
GTATTCTGAA
GTTTAGACAC
TTTTTTCCTT
CTTGGCCCAT
CGGATTTATG
CGGATTTAGT
TGGGACAAAA
TGATTACCAA
TTTTATTGTT
CCCCAGTGGA
AGGTGGCCGA
GGGCGGGGGT
TTAGTCAGTT
TGGGGGGAGT
TAATCGAAAA
TAATTGTGGA
P.AGCCATTGT
PGGCGATCGC
CCCTGCAGGA
CCTTTGCGGC
rGTGGGTAGC
%IAATTGCAGC
CCCATAGCTG
PGCCCGCCAC
~CTCTTTTTT
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 96/21022 WO 96/ 1022PCTIB95/O1 167 -46-
GGTTTAGCAT
TTGTCTATGT
AAGCTCAAAA
TGCAAAAAAG
CAGGCATCTG
TAACTCCCCC
ATGACTCACT
TTTAGTCTCC
TTTATCTATT
GGGGGGATGG
TTAGTATTTT
AGTAGCAAAA
TCAGATAAAA
CTCTAGGGAG
ATTTTTAGGC
GTAGAAGGCA
CCCGGCGCTG
TAAATTTATA
AACTCTTGAC
TAAGTTAACC
TAAGTTTAAT
TAAAAGCTTC
TTTTTCCGCT
AAAATCATAT
GACTAAAATT
GAGTTTTTTT
TCGGCCCAAT
AACAGCAGAG
TCATAACTGA
ACTTCGGTTT
GCCTTTAGAG
ACAGACTATC
CTAGCAATGG
GTAGTTAATG
GGTGATCAAG
GATAACTTCC
GTTTTACTGC
TATATTGTGA
AGTATTTTCT
CCAATATTGC
ACTCCCAGTT
GCGGTATAAT
AAAGAACGCT
AAAAGAAATT
TAAACAGCGG
CCATGGTTCC
CCAAGTCGGC
CAGAGCTTTG
GGAATAAATT
GTGAAAGTTT
A ATG Met CTA ACA GCG GAA AGA ATT AAA TTT ACC Leu Thr Ala Giu Arg Ile Lys Phe Thr CAG AAA CGG GGG Gin Lys Arg Gly CGT CGG GTA CTA Arg Arg Val Leu AAC CAA CGG Asn Gin Arg GTG GAT Val Asp GCC TAC Al a Tyr TTT GCC GAG Phe Ala Glu AAA ACC CTG Lys Thr Leu CTT TTT GCT Leu Phe Ala
CAT
His
ATT
Ile CTG ACC CAA Leu Thr Gln
GAT
Asp
TTT
Phe ATT GTG CTC Ile Val Leu
TGG
Trp s0
CCG
Pro AAT CCC TCC ATG TAT CTG Asn Pro Ser Met Tyr Leu TCC GCT TGG GCC TTT GTG Ser Ala Trp Ala Phe Val CTA CTG GGT TGT ATG OTT Leu Leu Gly Cys Met Val 1560 1620 1680 1740 1800 1860 1920 1980 2031 2079 2127 2175 2223 2271 2319 2367 2415 2463 CCA GTT ATT Pro Vai Ile TTG GCG Leu Ala
TTT
Phe 65
GCC
Ala GTG CGC Vai Arg ATC GCC TTG Ile Ala Leu TTT TCC TTC Phe Ser Phe GTC GGC CAC GAT Val Gly His Asp
AAC
Asn
GCC
Al a CAC AAT GCC His Asn Ala
TAT
Tyr
TTT
Phe TCC TCC AAT CCC Ser Ser Asn Pro
CAC
His 100
AGT
Ser AAC CGG GTT Asn Arg Val CTG GGC Leu Gly 105 ATG ACC TAC Met Thr Tyr CAC AAC TAT His Asn Tyr 125 GAA ATC CAT
GAT
Asp 110
TTG
Leu GTC GGG TTA Val Gly Leu
TCT
Ser 115
ACC
Thr TTT CTT TGG Phe Leu Trp CGC TAT CGC Arg Tyr Arg 120 CAT GAC GTG His Asp Val CAC CAC ACC His His Thr
TAC
Tyr 130
GTA
AAT ATT CTT Asn Ile Leu
GGC
Gly 135 GGA GAT GGC GCA CGT ATG AGT CCT Glu Ile 140 His Gly Asp Gly Ala Val Arg Met Ser 145 GAA CAA GAA CAT Glu Gin Glu His Pro 150 WO 96/21022 WO 9621022PCTIB9511167 -47-
GTT
Val 155 GGT ATT TAT CGT Giy Ile Tyr Arg
TTC
Phe 160 CAG CAA TTT TAT Gin Gin Phe Tyr
ATT
Ile 165 TOG GGT TTA TAT Trp Giy Leu Tyr
CTT
Leu 170 1TC ATT CCC TTT Phe Ile Pro Phe TOO TTT CTC TAC Trp Phe Leu Tyr
GAT
Asp 180 GTC TAC CTA OTG Vai Tyr Leu Val CTT AAT Leu Asn 185 AAA GGC AAA Lys Gly Lys TTA GCT AGT Leu Ala Ser 205
TAT
Tyr 190 CAC GAC CAT AAA His Asp His Lys
ATT
Ile 195 CCT CCT TTC CAG Pro Pro Phe Gin CCC CTA GAA Pro Leu Oiu 200 TAC OTT TTC Tyr Vai Phe TTO CTA 000 ATT Leu Leu Oly Ilie
AAO
Lys 210 CTA TTA TOO CTC Leu Leu Trp Leu
GOC
Gly 215 GOC TTA Giy Leu 220 CCT CTG OCT CTO Pro Leu Ala Leu
GGC
Oly 225 TTT TCC ATT CCT Phe Ser Ile Pro
GAA
Oiu 230 OTA TTA ATT GOT Val Leu Ile Gly
OCT
Ala 235 TCG OTA ACC TAT Ser Val Thr Tyr
ATO
Met 240 ACC TAT OOC ATC Thr Tyr Giy Ile
OTO
Val1 245 OTT TOC ACC ATC Val Cys Thr Ile
TTT
Phe 250 ATO CTG 0CC CAT Met Leu Ala His
OTO
Val 255 TTO GAA TCA ACT Leu Oiu Ser Thr
GAA
Oiu 260 TTT CTC ACC CCC Phe Leu Thr Pro OAT OGT Asp Giy 265 2511 2559 2607 2655 2703 2751 2799 2847 2895 2943 2991 3039 3088 3148 3208 OAA TCC GOT Oiu Ser Gly ACO 0CC AAT Thr Ala Asn 285 0CC Ala 270 ATT OAT GAC GAO Ile Asp Asp Oiu
TG
Trp 275 OCT ATT TOC CAA Ala Ile Cys Gin ATT COT ACC Ile Arg Thr 280 TTT TOT GOC Phe Cys Oly TTT 0CC ACC AAT Phe Ala Thr Asn
AAT
Asn 290 CCC TTT TOG AAC Pro Phe Trp Asn GOT TTA Gly Leu 300 AAT CAC CAA OTT Asn His Oin Val
ACC
Thr 305 CAC CAT CTT TTC His His Leu Phe
CCC
Pro 310 AAT ATT TOT CAT Asn Ile Cys His
ATT
Ile 315 CAC TAT CCC CAA His Tyr Pro Gin OAA AAT ATT ATT Olu Asn Ile Ile
AAO,
Lys 325 OAT OTT TOC CAA Asp Val Cys Oln TTT GOT OTO GAA Phe Gly Val Giu
TAT
Tyr 335 AAA OTT TAT CCC Lys Val Tyr Pro
ACC
Thr 340 TTC AAA OCO OCO Phe Lys Ala Ala ATC 0CC Ile Ala 345 TCT AAC TAT Ser Asn Tyr
CGC
Arg 350 TOG CTA GAO 0CC Trp Leu Oiu Ala
ATO
Met 355 OOC AAA OCA TCO Gly Lye Ala.Ser
TOACATTGCC
360 TTGGGATTGA AGCAAAATOO CAAAATCCCT COTAAATCTA TGATCOAAOC CTTTCTOTTG CCCGCCGACC AAATCCCCOA TGCTOACCAA AGOTTOATGT TOOCATTOCT CCAAACCCAC WO 96/21022 WO 96/ 1022PCTIB95O1 167 -48- TTTGAGGGGG TTCATTGGCC GCAGTTTCAA
GCTGACCTAG
TTGCTcAAT CCGCTGGGAT ATTGAAAGGC
TTCACCACCT
TGGGAAGGAC AAACCGTCAG AATTGTTTAT
TCTGGTGACA
TGGTCTAACC CAGCCCTGGC CAAGGCTTGG
ACCAAGGCCA
AGGCCAGAAA AATTATATTG GCTCCTGATT
TCTTCCGGCT
AGCATTTTTG CCAAGGAJATT CTATCCCCAC
TATCTCCATC
AATTTTATCC
ATCAGCTAGC
INFORMATION FOR SEQ ID NO:2: Ci SEQUENCE
CHARACTERISTICS:
LENGTH: 359 amino acids TYPE: amino acid TOPOLOGY: linear
GAGGCAAAGA
TTGGTTTCTA
CCATCACCCA
TGCAAATTCT
ATCGCACCTA.
CCACTCCCCC
TTGGGTGATT
CCCTGCTCAA
CCCATCCATG
CCACGAGGCT
CCGATTTTTG
GCCTGTAcAA 3268 3328 3388 3448 3508 3568 3588 (ii) MOLECULE (xi) SEQUENCE Leu Thr Ala Giu 5 Met 1 TYPE: protein DESCRIPTION: SEQ ID Arg Ile Lys Phe Thr 10 Arg Val Asp Ala Tyr NO: 2: Gin Lys Phe Ala Arg Gly Phe Arg Giu His Giy Leu Arg Vai Leu Asn Gin Thr Gin Arg Asp Asn Leu Trp Leu Phe Ser Pro Ser Met 40 Ala Trp Ala 55 Leu Gly Cys Phe Pro Tyr Leu Lys Thr Phe Val Leu Phe Met Val Leu Ala Leu Ala Ile Ile Val Pro Val Ile Val Arg Leu Al a Ile Ala Leu Phe Ser Phe Asn Ile 70 Val Asn Ser Asn Pro Gly Leu Ser 115 His 100 Ser Gly His Asp Ala 90 Arg Val Leu Gly 105 Trp Arg Tyr Arg Asn His Asn Ala Tyr Ser Met Thr Tyr His Asn Tyr Asp Phe Val 110 Leu His His Phe Leu 120 Thr Tyr Thr Asn Ile 130 Ala Val Arg Met Ser 145 Leu Gly His Asp Val Giu Ile His Gly Asp Gly 135 140 Pro Giu Gin Glu His Val Gly Ile Tyr Arg Phe 150 155 160 WO 96/21022 PCT/IB95/01167 -49- Gln Gin Phe Tyr lie Trp Gly Leu Tyr Leu Phe Ile Pro Phe Tyr Trp 165 170 175 Phe Leu Tyr Asp Val Tyr Leu Val Leu Asn Lys Gly Lys Tyr His Asp 180 185 190 His Lys Ile Pro Pro Phe Gin Pro Leu Glu Leu Ala Ser Leu Leu Gly 195 200 205 lie Lys Leu Leu Trp Leu Gly Tyr Val Phe Gly Leu Pro Leu Ala Leu 210 215 220 Gly Phe Ser Ile Pro Glu Val Leu Ile Gly Ala Ser Val Thr Tyr Met 225 230 235 240 Thr Tyr Gly Ile Val Val Cys Thr Ile Phe Met Leu Ala His Val Leu 245 250 255 Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly Glu Ser Gly Ala Ile Asp 260 265 270 Asp Glu Trp Ala Ile Cys Gin Ile Arg Thr Thr Ala Asn Phe Ala Thr 275 280 285 Asn Asn Pro Phe Trp Asn Trp Phe Cys Gly Gly Leu Asn His Gin Val 290 295 300 Thr His His Leu Phe Pro Asn Ile Cys His Ile His Tyr Pro Gin Leu 305 310 315 320 Glu Asn Ile Ile Lys Asp Val Cys Gin Glu Phe Gly Val Glu Tyr Lys 325 330 335 Val Tyr Pro Thr Phe Lys Ala Ala Ile Ala Ser Asn Tyr Arg Trp Leu 340 345 350 Glu Ala Met Gly Lys Ala Ser 355 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1884 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGCTTCACTT CGGTTTTATA TTGTGACCAT GGTTCCCAGG CATCTGCTCT AGGGAGTTTT TCCGCTGCCT TTAGAGAGTA TTTTCTCCAA GTCGGCTAAC TCCCCCATTT TTAGGCAAAA 120 WO 96/21022 WO 96/ 1022PCT/1EB95/01 167 TCATATACAG ACTATCCCAA TATTGCCAGA GCTTTGATGA CTCACTGTAG AAGGCAGACT 180
AAAATTCTAG
TTTTTTGTAG
CTAACAGCGG
CGGGTGGATG
CTGAAAACCC
CCAGTTATTT
TTTTCCTTCA
AACCGGGTTC
CGCCACAACT
GGAGATGGCG
CAATTTTATA
TACCTAGTGC
GAATTAGCTA
CTGGCTCTGG
TATGGCATCG
CTCACCCCCG
ACCACGGCCA
CACCAAGTTA
AATATTATTA
AAAGCGGCGA
TGCCTTGGGA
GTTGCCCGCC
CCACTTTGAG
GATTTTGCTC
TCAATGGGAA
CATGTGGTCT
CAATGGACTC CCAGTTGGAA
TTAATGGCGG
AAAGAATTAA
CCTACTTTGC
TGATTATTGT
TTCCGGTGCG
ATGTCGGCCA
TGGGCATGAC
ATTTGC-ACCA
CAGTACGTAT
TTTGGGGTTT
TTAATAAAGG
GTTTGCTAGG
GCTTTTCCAT
TGGTTTGCAC
ATGGTGAATC
ATTTTGCCAC
CCCACCATCT
AGGATGTTTG
TCGCCTCTAA
TTGAAGCAAA
GACCAAATCC
GGGGTTCATT
AAATCCGCTG
GGACAAACCG
AACCCAGCCC
TATAATGTGA
ATTTACCCAG
CGAGCATGGC
GCTCTGGTTG
CCTACTGGGT
CGATGCCAAC
CTACGATTTT
CACCTACACC
GAGTCCTGAA
ATATCTTTTC
CAAATATCAC
GATTAAGCTA
TCCTGAAGTA
CATCTTTATG
CGGTGCCATT
CAATAATCCC
TTTCCCCAAT
CCAAGAGTTT
CTATCGCTGG
ATGGCAAAAT
CCGATGCTGA
GGCCGCAGTT
GGATATTGAA
TCAGAATTGT
TGGCCAAGGC
TAAATTTTA
AAGTTTTTA
AAACGGGGGT
CTGACCCAAA
TTTTCCGCTT
TGTATGGTTT
CACAATGCCT
GTCGGGTTAT
AATATTCTTG
CAAGAACATG
ATTCCCTTTT
GACCATAAAA
TTATGGCTCG
TTAATTGGTG
CTGGCCCATG
GATGACGAGT
TTTGGAACT
ATTTGTCATA
GGTGTGGAAT
CTAGAGGCCA
CCCTCGTAAA
CCAAAGGTTG
TCAAGCTGAC
AGGCTTCACC
TTATTCTGGT
TTGGACCAAG
GTCTCCCCCG
TCTATTTAAA
TTCGTCGGGT
GGGATAATCC
GGGCCTTTGT
TGGCGATCGC
ATTCCTCCAA
CTAGTTTTCT
GCCATGACGT
TTGGTATTTA
ATTGGTTTCT
TTCCTCCTTT
GCTACGT
CTTCGGTAAC
TGTTGGAATC
GGGCTATTTG
GGTTTTGTGG
TTCACTATCC
ATAAAGTTTA
TGGGCAAAGC
TCTATGATCG
ATGTTGGCAT
CTAGGAGGCA
ACCTTTGGTT
GACACCATCA
GCCATGCAAA
GCGCTGGAGT
TTTATAAATG
ACTAAACCAA
CTCCATGTAT
GCTTTTTGCT
CTTGGCGGCC
TCCCCACATC
TTGGCGCTAT
GGAAATCCAT
TCGTTTCCAG
CTACGATGTC
CCAGCCCCTA
CGGCTTACCT
CTATATGACC
AACTGAATTT
CCAAATTCGT
CGGTTTAAAT
CCAATTGGAA
TCCCACCTTC
ATCGTGACAT
AAGCCTTTCT
TGCTCCAAAC
AAGATTGGGT
TCTACCCTGC
CCGACCCATC
TTCTCCACGA
240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 GGCTAGGCCA GAAAAATTAT ATTGGCTCCT GATTTCTTCC GGCTATCGCA CCTACCGATT WO 96/21022 WO 96/ 1022PCT/IB95/O1 167 -51- TTTGAGCATT TTTGCCAAGG AATTCTATCC CCACTATCTC CATCCCACTC
CCCCGCCTGT
ACAAAATTTT ATCCATCAGC
TAGC
INFORMATION FOR SEQ ID NO:4: SEQUENCE
CHARACTERISTICS:
LENGTH: 1685 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 1860 1884
AATATCTGCC
GAAATACATTl
GATTCAAGGG
TCCCTTGAAG
CTCTACATGG
TTCTGAGGTT
TGACAAAAAA
GAGTGTTTAT
GATGGGGTTT
AGTGTCTGAT
AATAAGTATT
TGAATATGAC
TTCACTCACC
TGTAAGTTAT
TGTACAATCT
CTTGGGATGC
GGGTGAAAGA
GTTCTCCTTG
GTTTGAGAAA
TACCCTCCCA
ACCTCAGATG
AAAGCCTATG
AGTCTTGCTG
AAGAATCTTG
TCTAAAGATT
GGTCATATTA
GGGGTTTTGT
CTTTGGATTC
TCAAGGCTTA
GGTTGGTGGA
CCTGATTTAC
TCTCATTTCT
CAACATTGGA
CTCATAATGT
CTAGTGTTCT
ATTATGTTTG
AACCACTTCT
CAAACGGATG
AAGAGAGTAG
AACTCAAGAA
ATGTTTCGGA
GTCAAGAGGT
ATA.AGTTTTT
ATAGGAAGCT
TGTTTGCAAC
TTTGTGAGGG
AGAGTGGTTG
ATAAGTTTAT
AATGGAACCA
AATATATACC
ATGAGAAAAG
CATTTTACCC
TGTTGACCAA
CGATTTGGTA
TTATTGCAAG
CTTCAAGTGT
GGACACTTGA
TCATTTTTCA
CCACGATAAJA
TTGGGTGAAA
AACTGATGCA
CACTGGGTAT
TGTGTTTGAG
TTTGTGCTTT
TGTTTTGGTA
GATTGGACAT
GGGTATTTTT
TAATGCACAT
ATTCCTTGTT
GTTGACTTTT
I
TATTATGTGT
GAGAAATGTG
CCCGTTGCTT
rTTATCAGTG
TTATGTTGGA
CATTTCTTGT C
TCAATGGCTG
CCCGGAGATC
GACCATCCAG
TTTGTTGCAT
TATCTTAAAG
TTTTCTAAAA
ATAGCAATGC
CATTTGTTTT
GATGCTGGGC
GCTGCAAATT
CACATTGCCT
GTGTCTTCCA
GACTCTTTAT
GCTGCTAGGC
rCCTATCGAG 3TTTCTTGTT kCTGGAATGC
AGCCTAAAG
CTCAAATcAA
TATGGATCTC
GTGGCAGCTT
TCCATCCTGC
ATTACTCTGT
TGGGTTTGTA
TGTTTGCTAT
CTGGGTGTTT
ATTATATGGT
GTCTTTCAGG
GTAATAGCCT
AGTTTTTTGG
CAAGATTCTT
TCAATATGTA
CTCAGGAACT
rGCCTAATTG kACAAGTTCA 3,GAATAATTG 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 TCATGGTGGA TTGCAATTCC AAATTGAGCA TCATTTGTTT CCCAAGATGC
CTAGATGCAA
1200 WO 96/21022 WO 9621022PCTLB95/01 167 -52-
CCTTAGGAAA
TTATGCATCT
GCAGGCTAGG
TCATGGTTAA
GTGTCTTGTC
ATGTTTTTTA
TGCATATTGT
TGTTTTCAGT
TATTT
ATCTCGCCCT
TTCTCCAAGG
GATATAACCA
AATTACCCTT
TTGGTTCTAC
ATATATTTTA
CAATTGTTGT
TGAAGCTCAT
ACGTGATCGA
CCAATGAAAT
AGCCGCTCCC
AGTTCATGTA
TTGTTGGAGT
GAGGTTTTGC
GCTCAATATC
GTGTACTTCT
GTTATGCAAG
GACACTCAGA
GAAGAATTTG
ATAATTTGAG
CATTGCAACT
TTTCATCTCC
TGATATTTTG
ATAGACTTTG
AAACATAATT
ACATTGAGGA
GTATGGGAAG
ATTATGTATC
TGTCTTTTAT
ATTATTGATG
GAATGTACTT
TTTAAATGGT
TGCCTTACAA
ACACAGCATT
CTCTTCACAC
TCCTATGTTT
GGTTTATTAG
AATAAGGAGT
TGTACCACTG
TATGTCATGT
1260 1320 1380 1440 1500 1560 1620 1680 1685 (2) INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 448 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Ala Gin Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu 1 His Asp 10 Ser Lys Asn Asp Lye Pro Val Ser Asp Gly Asp Leu Trp Ile 25 His Ile Gin Gly Trp Val Lys Lys Ser Leu Asp 40 Val Pro Gly Gly Ser Val1 Lye Ala Tyr Phe Pro Leu Ala Phe His Ala Gly Gin Pro Ala Glu 55 Asn Thr Asp Ala Phe Phe Ser Thr Trp Leu Lys 70 Val Leu Asp Lys Phe 75 Lys Thr Giy Tyr Tyr Lye Asp Tyr Ser Rpr Ser Glu Val Ser 90 Tyr Asp Tyr Arg Lys Leu Val Phe Glu Met Phe Ala 115 Tyr Gly Vai 130 Phe 100 Thr Lys Met Gly Leu 105 Ala Asp Lye Lye Leu Cys Phe Phe Cys Giu 135 Ile 120 Gly Met Leu Phe Ala 125 Leu Gly His Ile 110 Met Ser Val Phe Ser Gly Leu Val Leu Val WO 96/21022 WO 9621022PCTIIIB95/01 167 -53- Cys 145 Ala Gly Lys Asp Phe 225 Ser Ile Leu Cys Asn 305 Gly Tyr Gly Gly Cys 385 His Thr Lys Leu Gly Ile Trp Pro 210 Gly Leu Met Leu Leu 290 Trp Met Val Thr Ser 370 Asn Asn Leu Pro Met His Phe Asn 195 Asp Ser Ser Cys Thr 275 Val Gly Gin Giy Leu 355 Gin Leu Leu Arg Leu 435 Gly Tyr Ala 180 His Leu Leu Arg Ala 260 Lys Phe Giu Gin Lys 340 Asp Phe Arg Pro Thr 420 Pro Phe Met 165 Ala Asn Gin Thr Phe 245 Ala Arg Ser Arg Vai 325 Pro Ile Gin Lys Tyr 405 Leu Lys Leu 150 Vai Asn Ala Tyr Ser 230 Phe Arg Asn Ile Ile 310 Gin Lys Ser Ile Ile 390 Asn Arg Asn Trp Val Cys His Ile 215 His Val1 Leu Val Trp 295 Met Phe Giy Cys Giu 375 Ser Tyr Asn Leu Ile Ser Leu His 200 Pro Phe Ser Asn Ser 280 Tyr Phe Ser Asn Pro 360 His Pro Ala Thr Val 440 Gin Asp Ser 185 Ile Phe Tyr Tyr Met 265 Tyr Pro Val Leu Asn 345 Pro His Tyr Ser Ala 425 Trp Ser Gly 155 Ser Arg 170 Giy Ile Ala Cys Leu Val Giu Lys 235 Gin His 250 Tyr Val Arg Ala Leu Leu Ile Ala 315 Asn His 330 Trp Phe Trp Met Leu Phe Val Ile 395 Phe Ser 410 Leu Gin Giu Ala Trp Leu Ser Asn Val1 220 Arg Trp Gin Gin Val1 300 Ser Phe Giu Asp Pro 380 Giu Lys Ala Leu Ile Asn Ile Ser 205 Ser Leu Thr Ser Giu 285 Ser Leu Ser Lys Trp 365 Lys Leu Al a Arg His 445 Gly Lys Gly 190 Leu Ser Thr Phe Leu 270 Leu Cys Ser Ser Gin 350 Phe Met Cys Asn Asp 430 Thr His Phe 175 Trp Giu Lys Phe Tyr 255 Ile Leu Leu Val Ser 335 Thr His Pro Lys Giu 415 Ile His Asp 160 Met Trp Tyr Phe Asp 240 Pro Met Gly Pro Thr 320 Val Asp Gly Arg Lys 400 Met Thr Gly WO 96/21022 PCT/IB95/01167 -54- INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Trp Ile Gly His Asp Ala Gly His 1 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asn Val Gly His Asp Ala Asn His 1 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Val Leu Gly His Asp Cys Gly His 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: WO 96/21022 PCT/IB95/01167 Val Ile Ala His Glu Cys Gly His 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Val Ile Gly His Asp Cys Ala His 1 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Val Val Gly His Asp Cys Gly His 1 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: His Asn Ala His His 1 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid.
TOPOLOGY: linear WO 96/21022 PCT/IB95/01167 -56- (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: His Asn Tyr Leu His His 1 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: His Arg Thr His His 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID His Arg Arg His His 1 WO 96/21022 PCT/IB95/01167 -57- INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: His Asp Arg His His 1 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: His Asp Gin His His 1 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: His Asp His His His 1 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 96/21022 PCT/IB95/01167 -58- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: His Asn His His His 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Phe Gin Ile Glu His His 1 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: His Gin Val Thr His His 1 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: His Val Ile His His 1 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid WO 96/21022 PCT/IB95/01167 -59- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: His Val Ala His His 1 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: His Ile Pro His His 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID His Val Pro His His 1

Claims (21)

1. An isolated nucleic acid encoding a A-6 desaturase from Borago officinalis comprising the nucleotide sequence of SEQ ID NO: 4.
2. An isolated nucleic acid that codes for the amino acid sequence of SEQ ID NO:
3. A vector comprising the nucleic acid of any one of Claims 1 2.
4. An expression vector comprising the isolated nucleic acid of any one of Claims 1 2 operably linked to a promoter and optionally a termination signal capable of effecting expression of the gene product of said isolated nucleic acid. The expression vector of Claim 4 wherein said promoter is a A-6 desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycinin promoter, a napin promoter, the 35S promoter from CaMV, or a helianthinin tissue-specific promoter.
6. The expression vector of Claim 4 wherein said promoter is constitutive or tissue-specific.
7. The expression vector of Claim 4 wherein said termination signal is a Synechocystis termination signal, a nopaline synthase termination signal, or a seed termination signal.
8. A cell comprising the vector of any one of Claims 3 7.
9. The cell of Claim 8 wherein said cell is an animal cell, a bacterial cell, a plant cell or a fungal cell. An organism genetically transformed with the isolated nucleic acid of any one of Claims 1 2.
11. An organism genetically transformed with the vector of any one of Claims 3 7.
12. The organism of Claim 10 or 11 wherein said organism is a bacterium, a fungus, a 0 plant or an animal. Sc:\mipatext\abilang rhonea.doc 61
13. A plant or progeny of said plant which has been regenerated from the plant cell of Claim 9 and wherein said plant or said progeny of said plant is genetically transformed with the nucleic acid of any one of Claims 1 2.
14. The plant of Claim 13 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises: transforming a plant cell with the isolated nucleic acid of any one of Claims 1 2; and regenerating a plant with increased GLA content from said plant cell.
16. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises: transforming a plant cell with the vector of any one of Claims 3 7; and regenerating a plant with increased GLA content from said plant cell.
17. The method of Claim 15 or 16 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.
18. A method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA which comprises transforming said organism with the isolated nucleic acid of any one of Claims 1 2.
19. A method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA which comprises transforming said organism with the vector of any one of Claims 3 7. A method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA and linolenic acid (LA) which comprises transforming said organism with the isolated nucleic acid of any one of Claims 1 or 2 and an isolated nucleic acid encoding Al2-desaturase.
21. The method of Claim 20 wherein said isolated nucleic acid encoding /6-desaturase comprises nucleotides 44 to 1390 of SEQ. ID NO: 4. Ix£
22. A method of inducing production of octadecatetraeonic acid in an organism deficient or lacking in gamma linolenic acid which comprises transforming said organism with the isolated nucleic acid of any one of Claims 1 2.
23. A method of inducing production of octadecatetraeonic acid in an organism deficient or lacking in gamma linolenic acid which comprises transforming said organism with the vector of any one of Claims 3 7.
24. The method of Claim 22 or 23 wherein said organism is a bacterium, a fungus, a plant or an animal. A method of producing a plant with improved chilling resistance which comprises: transforming a plant cell with the isolated nucleic acid of any one of Claims 1 2; and regenerating said plant with improved chilling resistance from said transformed plant cell.
26. The method of Claim 25 wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.
AU46735/96A 1994-12-30 1995-12-28 Production of gamma linolenic acid by a delta6-desaturase Ceased AU707061B2 (en)

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US08/366,779 US5614393A (en) 1991-10-10 1994-12-30 Production of γ-linolenic acid by a Δ6-desaturase
PCT/IB1995/001167 WO1996021022A2 (en) 1994-12-30 1995-12-28 Production of gamma linolenic acid by a δ6-desaturase

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US5614393A (en) 1997-03-25
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